This collaborative proposal between the University of North Carolina Chapel Hill and Emory University is an adjunct pilot metabolomic study to an ongoing multi-center, randomized, double-blind, placebo-controlled, modified 2 x 2 factorial chemoprevention clinical trial (R01 CA98286; PI: J. Baron) (n = 2,259) that is testing the efficacy of supplemental vitamin D3 (1,000 IU daily) and calcium (1,200 mg elemental calcium daily), alone and in combination vs. placebo over 3 5 years in preventing sporadic colorectal adenoma recurrence (the parent study). The objective of this proposal is to establish a new interactive collaboration between clinical researchers in the parent study and metabolomics experts, and to incorporate a novel high-resolution metabolomics approach into the parent study to better understand the independent and synergistic antineoplastic actions and other effects of vitamin D and calcium in humans. Our innovative approach utilizes high-resolution mass spectrometry to measure >20,000 metabolites, and provides a unique workflow using false discovery rates (FDR) to prioritize metabolites for subsequent study, correlation analysis to enhance identification of relevant metabolic modules associated with these prioritized metabolites, pathway mapping using available online tools to identify relevant metabolic pathways, and post-hoc application of ion dissociation (MS/MS) spectroscopy to representative metabolites to confirm pathway identification. In the pilot study we will obtain preliminary data on the effects of supplementation with vitamin D and/or calcium on individual metabolites and metabolic pathways in a sub-set of participants (n = 120) with baseline and follow-up biopsies of normal-appearing rectal mucosa. The preliminary results obtained from this pilot study will lead to larger, full-scale, metabolomic investigations to elucidate 1) the individual and combined effects of vitamin D and calcium on metabolomic pathways in humans (thus providing a more comprehensive assessment of the benefits and risks of these heavily promoted agents as supplements), 2) whether these metabolomic changes correlate with genetic variation and systemic and tissue-specific biomarkers in humans (and thus their direct relevance to the chemoprevention of colorectal neoplasms), and 3) whether the metabolomic changes predict the occurrence of sporadic colorectal adenomas in humans, thus paving the way for the development of treatable biomarkers of risk for colorectal neoplasia that are analogous to lipid profiles for the prevention of ischemic heart disease. Elucidating the effects of vitamin D and/or calcium supplementation on individual metabolites and metabolomic pathways may inform the potential use of these chemopreventive agents for preventing colorectal neoplasms, and, possibly, other chronic diseases.